ELSEVIER Synthetic Metals 102 (1999) 1328-1329

A polypyrrole-biotin based biosensor: elaboration and characterization

L. M. Torres-Rodriguez, M. Billon, A. Roget and G. Bidan Laboratoire dElectrochimie Moldculaire. CllMR 5819 CEA-CNRS-Universitt Joseph Fourier, Dipartement de recherche fondamentale

sur la Mati&e Condenske, CEA Grenoble, I7 Av. des Martyrs, 38054 Grenoble Ckdex 09 France

Abstract In this paper, we present the electrosynthesis and the electrochemical characterization of a polypyn-ole including biotin units in

order to realize a versatile avidin biosensors. The accessibility of immobilized biotin entities toward avidin conjugates has been shown by the quarzt crystal microbalance.

Keywords: Electrochemical polymerization, Polypyrrole and derivates, Biosensors, Biotin.

1. Introduction The elaboration of enzyme sensors has been a topic of

considerable interest in recent years due to their potential applications [ 11. Their fabrication needed the immobilization of enzymes on the electrode surface. Several techniques have been developed such as (i) the anchoring of biotinylated enzymes to the avidin-modified electrode thanks to the strong affinity between avidin and biotin [2, 31 or (ii) the insertion of enzymes within an electronic conducting polymer (ECP) matrix by entrapment during its electropolymerization [4].

We report herein a novel strategy based on copolymerization of pyrrole monomers and pyrrole covalently linked to biotin through a spacer arm (Scheme 1).

spacer

\

,,i,,i

~~~z~,~~~~~z~zlz~~~~z~~~~~~~~~~~~ Jj s i --------7 I I Q 1

111; O\

_______________________ z

I _ - - _ _ - _ _ _ Biotin pyrrole unit

Scheme 1 : The pyrrole-biotin 1.

The synthetic strategy leads (reaction 1, scheme 2) to an ECP film anchored on the electrode surface where biotin entities are grafted on the polypyrrole network. Then the immobilization on the film of biomolecules such as avidin enzyme conjugates, can be realized. Indeed these immobilized biotin entities appear as anchoring points since biomolecules bearing avidin units can be easily linked on the polypyrrole surface thanks to the biotimavidin interaction (reaction 2) and this in a one step only whitout any chemical coupling.

This approach shows several features. On the one hand, one

* Corresponding author (E-mail : billon@drfmc.ceng.cea.fr)

could elaborate a large number of biosensors due to a wide variety of commercial avidin conjugates and on the other hand it goes the possibility of the miniaturization thanks to the electrochemical addressing of the electropolymerization as we have previously shown for the polypyrrole DNA chip [5].

CoPoLY] I 1 (

l/r AVIDIN Scheme 2 : Synthetic strategy towards a avidin biosensor.

After the description of the electropolymerization and the electrochemical properties of the biotin polypyrrole, we present in this report the results achieved by quartz crystal microbalance (QCM) showing that the film provides biotin sites capable of selectively binding avidin.

2. Experimental Electrochemical syntheses of the biotinylated copolypyrrole

and their characterizations by cyclic voltamperometry were made with a PAR 273 potentiostat from EG&G Princeton Applied Research controlled by a computer. The working electrode was a platinum electrode (diameter, 3 mm). All potentials are relative

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M. Billon et al. I Synthetic Metals 102 (1999) 1328-1329 1329

to a saturated calomel electrode (SCE). The electrolyte was an aqueous solution containing 3% CHsCN with 0.1 mol L-i LiC104 (Fluka Purum) which was purged by argon bubbling before each experiment.

The quartz crystal is AT-cut with a basic resonance frequency of 9 Mhz and sandwiched between two Pt electrodes (diameter, 5 mm). The frequencies were measured by a Hp 53424 A frequency counter from Hewlett Packard.

3. Results and Discussion The pyrrole-biotin 1 was synthesised by coupling an amino

alkyl pyrrole and a biotm entity [6]. Concerning the electrosynthesis of the biotinylated copolypyrrole (noticed copoly[l]), it was realized by repeated potentiel linear scanning between -0.25 and +0.85 V from a solution containing 20 mM pyrrole and 10 mM pyrrole-biotin monomers 1. The cyclic voltammogram recorded during the electropolymerization shows two responses. One is an irreversible and sharper anodic peak due to monomer oxidation at approximatively +0.81 V and the other is constituted of an oxidation peak at +0.55 V and a reduction peak at -0.10 V. Both responses increased in intensity with subsequent scans.

ARer synthesis and thoroughy rinsing, the modified electrode is transferred to an electrolytic solution free of monomers. As shown Figure 1, the electrochemical response of this modified electrode is composed of one reversible and stable response with a shape typical of that observed with an ECP deposed on the surface electrode. In comparison with the electroactivity associated to an polypyrrole film, we observed that the response of copolyu] film is shifted to the more positive potential values and characterized by a higher value (340 mV) of AEp (AEp =Epc-Epa). And more, the electroactivity of the biotinylated copolypyrrole occurs in a large potential range in agremment with a mixed behaviour of poly(pyrrole) and poly(N-substitued pyrrole).

120

80

40

0

-40

-80

-120 1

E vs SCE

Fig. 1. Cyclic voltammetry of copoly[l] film on a platinum electrode (diameter 3 mm) in H20/LiC104 0.1 M (v = 50 mV/s). Synthesis of the film (see text) was performed by repeated potentiel linear scanning by passing 34 mC/cm.

anchor to the surface of the biotinylated copolypyrrole via the biotinlavidin interaction.

After the electrosynthesis on the platinum surface of the QCM, the copoly[l] film was placed in a aqueous solution containing the Denhardts reagent in order to avoid non-specitic adsorption of avidin. The response of QCM (Figure 2) before and after the injection of an avidin solution shows a fast decreasing of frequency just after the addition of avidin conjuguate. From this result, we have assessed that the mass of avidin immobilized on the the copoly[l] film equal to 48.03 ng cooresponding to 0.7 pmol of avidin conjugates immobilized on the copolyu] film.

FW

\ b

Fig. 2. Variation of frequency with the time of the copoly[l] film in aqueous solution containing the Denhardts reagent, before (a) and after (b) the injection of an avidin solution.

The same experiment has been realized with a polypyrrole film. In this case, the injection of avidin did not induced a revealing variation of the frequency in comparison with this observed for the biotinylated copolypyrrole.

4. Conclusion This study has demonstrated shows the possibility to

immobilize biotin units linked to the polypyrrole matrix. Although these biotin units were inserted first in the ECP, some of their were accessible on the polypyrrole surface to avidin conjugates. Thus the biotinylated polypyrrole can be use in order to elaborate a versatile avidin biosensor.

5. References [l] G. Bidan in Gabor Harsanyi (Ed.), Polymer films in sensor

applications, Ph. D. Gabor Harsanyi, Hungary, (1995) 206. [2] T. Hoshi, J.-I. Anzai, T. Osa, Anal. Chem., 67 (1995) 770. [3] M. Vreeke, P. Rocca, A. Heller, Anal. Chem., 67 (1995) 303. [4] P. Bartlett, J. Cooper, J. Electroanal. Chem., 362 (1993) 1. [5] T. Livache, B. Fouque, A. Roget, J. Marchand, G. Bidan, R.

Teoule, G. Mathis, Anal. Biochem., 255 (1998) 188. [6] L. M. Torres-Rodrigez, A. Roget, M. Billon, T. Livache, G.

Bidan, submitted for publication. In order to check the ability of recognition characteristic of

the copolyu] film towards avidin conjugates, a study by the quartz microbalance was undertaken. Indeed the great sensitivity of this device allows to determine the mass of avidin which can